Many Escherichia coli promoters are difficult to recognize by sequence alone. Numerous studies have shown that the optimal sigma70?dependent RNA polymerase binding site contains -35 and -10 consensus hexamers spaced 17 bp apart, though much deviation from this consensus is encountered with bona fide promoters. It can be estimated that there are at least 10 times as many such degenerate sites as there are ORFs in E. coli. Historically, most promoters were sought upstream of open reading frames (ORFs) or stable RNA genes, but their presence elsewhere has not been systematically investigated. In general, promoters were not expected to be found within ORFs though some internal promoters do exist within operons. We used a cloning approach to discover other potential promoters in E. coli. Random chromosomal fragments of ~160 bp were fused to a promoterless lacZ reporter gene on a multicopy plasmid. 105 such clones were sequenced and assayed for promoter activity. 56 had significant activity. For six of the nine most active sequences (with orientations opposite to that of the ORF), expression from the normal E. coli chromosome was detected by RT-PCR, but defined transcripts were not detected by Northern analysis. The activity of one of these sequences (studied in greater depth) could be enhanced by the presence of translational signals or UP elements (AT rich sequences upstream of the promoter). Our results indicate that the E. coli chromosome carries numerous -35 and -10 sequences with weak promoter activity but that most are not productively expressed because other features needed to enhance promoter activity and transcript stability are absent. An open question is whether these RNA polymerase binding sites and the accompanying low levels of intragenic transcription have a function. Perhaps the binding sites serve as docks for RNA polymerase or the low levels of transcription help to maintain a more accessible state of the chromosome.